CN113740534A - Novel coronavirus neutralizing antibody detection card and preparation method thereof - Google Patents
Novel coronavirus neutralizing antibody detection card and preparation method thereof Download PDFInfo
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Abstract
The invention relates to a novel coronavirus neutralizing antibody detection card, which comprises a PVC (polyvinyl chloride) gasket, and a sample pad, a fluorescent pad, a nitrocellulose membrane and a water absorption pad which are arranged on the upper surface of the PVC gasket and are sequentially connected; the fluorescent pad is provided with a novel coronavirus recombinant protein marked by a fluorescent microsphere and an independent quality control system marked by the fluorescent microsphere, wherein the independent quality control system is hemocyanin; the nitrocellulose membrane is provided with a detection area and a quality control area, the detection area is coated with ACE2 protein, and the quality control area is coated with an anti-hemocyanin antibody; the hemocyanin of the independent quality control system and the anti-hemocyanin antibody of the quality control area can be interchanged. The detection card provided by the invention has the advantages that the detection result of the quality control area is not influenced by the detection substance in the detection area, and the detection result in the detection area and the quality control area have no cross influence, so that the detection result of the novel coronavirus neutralizing antibody is more accurate and the repeatability is better.
Description
Technical Field
The invention relates to the field of immunodetection, in particular to a novel coronavirus neutralizing antibody detection card and a preparation method thereof.
Background
A novel coronavirus, also known as 2019-nCoV, is a virus that can cause respiratory diseases. This virus can cause inflammation and the accumulation of mucus and body fluids in the airways of the lungs (pneumonia). Coronaviruses are of various types, most of which only infect animals, but sometimes mutate and infect humans.
The new coronavirus pneumonia epidemic situation becomes a global important public health event, and multiple experts think that the final control of global new corona epidemic depends on new corona vaccination and the organism to generate an antibody with high-efficiency immunity to the new coronavirus, namely a neutralizing antibody.
At present, the gold test standard for detecting neutralizing antibodies requires the treatment of new live coronaviruses in a biosafety tertiary protection laboratory, and is time-consuming, usually taking 2 to 4 days to complete. Virus neutralization assays based on pseudoviruses are considered to be another method of detecting neutralizing antibodies that can be performed in biosafety secondary laboratories, but still require the use of live viruses and cells. At present, a detection card detection mode based on an immunochromatography technology is also adopted, a quality control region (C) of the detection card is coated with a goat anti-mouse antibody, so that the deviation of a C line test value is large and the T/C interference is serious when different samples are tested due to the influence of HAMA (human anti-mouse antibody) in the samples, and the 2019-nCoV Spike Protein (RBD) marked by a fluorescent microsphere combined with the C line is the rest part which is not combined with ACE2, so that double interference is caused, and the repeatability is poor.
In order to better monitor infection rate, population immunity and protective immunity, and evaluate vaccine efficacy during clinical trials and after mass vaccination, a rapid test capable of stably and reliably detecting neutralizing antibodies against the novel coronavirus is urgently needed.
Disclosure of Invention
The invention provides a novel coronavirus neutralizing antibody detection card and a preparation method thereof, which are used for solving the problems of long time consumption, complex operation and unsuitability for large-scale detection in the prior art for detecting a neutralizing antibody and simultaneously overcoming the problems of large deviation and inaccuracy of detection results.
In order to achieve the purpose, the invention provides the following technical scheme: a novel coronavirus neutralizing antibody detection card comprises a PVC liner, and a sample pad, a fluorescent pad, a nitrocellulose membrane and a water absorption pad which are arranged on the PVC liner and sequentially connected; the fluorescent pad is provided with a novel coronavirus recombinant protein marked by a fluorescent microsphere and an independent quality control system marked by the fluorescent microsphere, wherein the independent quality control system is hemocyanin; the nitrocellulose membrane is provided with a detection area and a quality control area, the detection area is coated with ACE2 protein, and the quality control area is coated with an anti-hemocyanin antibody; the hemocyanin of the independent quality control system and the anti-hemocyanin antibody of the quality control area can be interchanged. The novel coronavirus recombinant protein is an S protein receptor binding domain.
Preferably, the nitrocellulose membrane is attached to the middle part of the PVC liner, the fluorescent pad and the water absorption pad are respectively lapped on the upper surfaces of two ends of the nitrocellulose membrane, the sample pad is lapped on the upper surface of the fluorescent pad, and the lapping and overlapping parts are all arranged at 1-3 mm.
Preferably, the solid content of the fluorescent microsphere is 0.1-5%, and the particle size is 100-300 nm.
Preferably, the width of the PVC liner is set to be 3-5 mm.
The invention further provides a preparation method of the novel coronavirus neutralizing antibody detection card, which comprises the following steps:
the method comprises the following steps: preparing a fluorescent pad and a sample pad, pretreating and drying the glass fiber or polyester film for later use;
step two: fluorescent labeling of novel coronavirus recombinant proteins;
step three: fluorescence labeling of the independent quality control system;
step four: uniformly mixing the novel coronavirus recombinant protein fluorescently labeled in the step two and the independent quality control system fluorescently labeled in the step three according to the proportion of 1:1 to obtain a fluorescent marker for later use;
step five: spraying the fluorescent marker of the step four on the fluorescent pad;
step six: respectively diluting an anti-hemocyanin antibody and ACE2 protein, and then coating the diluted anti-hemocyanin antibody and ACE2 protein on a nitrocellulose membrane to respectively form a quality control area and a detection area;
step seven: sticking the nitrocellulose plastic film formed in the sixth step to the middle of a PVC lining plate, lapping the fluorescent pad formed in the fifth step on one end of the nitrocellulose plastic film, lapping the sample pad formed in the first step on one end, far away from the nitrocellulose plastic film, of the fluorescent pad, and lapping the water absorption pad on the other end of the nitrocellulose plastic film;
step eight: and (6) cutting the test strip.
Wherein, in the second step, the fluorescence labeling of the novel coronavirus recombinant protein comprises the following steps:
the first process is as follows: cleaning microspheres, adding 100ul of fluorescent microspheres into 900ul of activating solution, blowing and uniformly mixing by using a pipette, centrifuging, removing supernatant, and adding 1ml of activating solution for later use;
and a second process: activating microspheres, adding 2.5ul EDC and 2.5ul NHS into the solution in the first process, activating at room temperature for 15min, centrifuging, removing supernatant, and adding 1ml of coupling solution for later use;
and a third process: coupling microspheres, adding 500ug of the novel coronavirus recombinant protein 200 into the solution in the process II, and reacting at room temperature for 1 h;
and (4) a fourth process: sealing, adding 100ul of sealing liquid into the solution in the third process, reacting for 1h at room temperature, centrifuging and removing supernatant;
and a fifth process: and (4) redissolving, namely adding 3-5ml of redissolving solution into the solution in the process four for redissolving.
In the third step, the fluorescence labeling independent quality control system comprises the following procedures:
the first process is as follows: cleaning microspheres, adding 100ul of fluorescent microspheres into 900ul of activating solution, blowing and uniformly mixing by using a pipette, centrifuging, removing supernatant, and adding 1ml of activating solution for later use;
and a second process: activating microspheres, adding 2.5ul EDC and 2.5ul NHS into the solution in the first process, activating at room temperature for 15min, centrifuging, removing supernatant, and adding 1ml of coupling solution for later use;
and a third process: coupling microspheres, adding 500ug of hemocyanin 200 into the solution in the process II, and reacting for 1h at room temperature;
and (4) a fourth process: sealing, adding 100ul of sealing liquid into the solution in the third process, reacting for 1h at room temperature, centrifuging and removing supernatant;
and a fifth process: and (4) redissolving, namely adding 3-5ml of redissolving solution into the solution in the process four for redissolving.
Preferably, in the fifth step, the fluorescent marker is sprayed on the fluorescent pad in an amount of 3-8 ul/cm.
Preferably, in the sixth step, the anti-hemocyanin antibody and the ACE2 protein are respectively diluted by using coating liquid, the anti-hemocyanin antibody is diluted to 0.3-1.0mg/ml, and the ACE2 protein is diluted to 0.8-1.2 mg/ml.
The invention further provides a novel coronavirus neutralizing antibody detection kit, which comprises the novel coronavirus neutralizing antibody detection card and a sample diluent, wherein the sample diluent comprises: Tris-HCl buffer solution with the pH value of 8.0-9.5, BSA with the mass concentration of 0.1-1% and Proclin300 with the mass concentration of 0.1-0.5%.
The invention has at least the following advantages and beneficial effects: the invention adopts an independent quality control system, the fluorescent pad is marked with hemocyanin/anti-hemocyanin antibodies, the quality control area is coated with the anti-hemocyanin antibodies/hemocyanin, the detection result of the quality control area is an independent quality control system combined by hemocyanin and anti-hemocyanin antibodies, the detection result is not influenced by the detection material of the detection area, the detection result of the detection area and the quality control area have no cross influence, so that the detection result of the novel coronavirus neutralizing antibodies is more accurate and has better repeatability, meanwhile, the aggregation of fluorescent materials in the quality control area is the standard for judging whether enough samples exist or not and the chromatography process is normal, and meanwhile, the fluorescent material aggregation can also be used as the internal control standard of a new coronavirus neutralizing antibody detection card.
Drawings
FIG. 1 is a test result reaction model of the test card of the present invention and the test card of the conventional art;
FIG. 2 is a front view of the test card of the present invention;
FIG. 3 is the accuracy analysis of the novel coronavirus neutralizing antibody detection kit provided by the invention, and the correlation analysis with the ELISA method.
In the figure, 1, an independent quality control system detects a card-positive result, 2, an independent quality control system detects a card-negative result, 3, a traditional detection card-negative result, 4, fluorescent microspheres, 5, S protein Receptor Binding Domain (RBD), 6, hemocyanin, 7, ACE2 protein, 8, anti-hemocyanin antibody, 9, neocoronaviruse neutralizing antibody, 10, goat anti-mouse antibody, 11, interferent, 12, PVC liner, 13, sample pad, 14, fluorescent pad, 15, nitrocellulose membrane, 16, water absorption pad, 17, quality control area and 18 detection area.
Detailed Description
Other advantages and features of the present invention will become readily apparent to those skilled in the art from the following detailed description, wherein it is to be understood that the invention is not limited to the specific embodiments disclosed, but is to be construed as limited only by the appended claims. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are commercially available products.
As shown in figures 1 and 2, the invention discloses a new coronavirus neutralizing antibody detection card, which comprises a PVC liner 12, and a sample pad 13, a fluorescent pad 14, a nitrocellulose membrane 15 and a water absorption pad 16 which are arranged on the upper surface of the PVC liner 12 and sequentially connected, wherein the nitrocellulose membrane 15 is specifically attached to the middle part of the PVC liner 12, the fluorescent pad 14 and the water absorption pad 16 are respectively lapped on the upper surfaces of two ends of the nitrocellulose membrane 15, the sample pad 13 is lapped on the upper surface of the fluorescent pad 14, and the lapping overlapped parts are all arranged at 1-3mm, so that the sample pad 13 and the fluorescent pad 14 are fully contacted, and a sample is controlled to be transmitted from the sample pad 13 to the fluorescent pad 14 at a proper speed while a chromatography process is ensured, so that the chromatography process is controlled, and a uniform chromatography effect is achieved. The sample pad 13 is used for bearing a sample and a sample buffer solution, the fluorescent pad 14 is used for bearing a compound formed by labeling proteins with fluorescent microspheres 4, the sample pad 13 and the fluorescent pad 14 can be made of glass fiber or polyester films, the material can ensure the absorption and release capacity of the sample and reduce the nonspecific adsorption of the sample pad 13 and the fluorescent pad 14 to the proteins in the sample, the nitrocellulose membrane 15 is used for coating a quality control area (C) 17 and a detection area (T) 18, the water absorption pad 16 is used for providing lateral chromatography power, the PVC liner 12 is used for connecting components on the upper surface of the PVC liner and providing support, the width is set to be 3-5mm, and the width setting ensures that sufficient waste liquid recovery capacity and recovery power are provided.
Specifically, a fluorescent microsphere 4-labeled novel coronavirus recombinant protein and a fluorescent microsphere 4-labeled independent quality control system are sprayed on the fluorescent pad 14, the novel coronavirus recombinant protein is an S protein Receptor Binding Domain (RBD) 5, and the independent quality control system is hemocyanin or an anti-hemocyanin antibody; the solid content of the fluorescent microsphere 4 is 0.1-5%, preferably 1%, and the particle size is 100-300 nm. The nitrocellulose membrane 15 is provided with a detection area 18 and a quality control area 17, the detection area 18 is coated with ACE2 protein 7, and the quality control area 17 is coated with an anti-hemocyanin antibody or hemocyanin; the label of the independent quality control system and the coating of the quality control area 17 can be interchanged, that is, when the independent quality control system is labeled as hemocyanin, the quality control area 17 is coated with an anti-hemocyanin antibody, and when the independent quality control system is labeled as an anti-hemocyanin antibody, the quality control area 17 is coated with hemocyanin.
The detection principle of the novel coronavirus neutralizing antibody detection card provided by the invention is as follows: the detection card adopts the principles of immunochromatography and competition method to detect the novel coronavirus neutralizing antibody in human serum, plasma and whole blood. The detection card contains RBD5 marked by fluorescent microspheres 4 and anti-hemocyanin antibodies marked by the fluorescent microspheres 4, which are coated on a fluorescent pad 14 in advance, and ACE2 protein 7 fixed on a detection area 18 and hemocyanin fixed on a quality control area 17 on a nitrocellulose membrane 15. During testing, when the sample does not contain the neutralizing antibody 9 of the new coronavirus or the content of the neutralizing antibody is lower than the minimum detection limit, the RBD5 marked by the fluorescent microspheres 4 coated on the fluorescent pad 14 in advance moves upwards under the capillary effect and is directly combined with ACE2 protein 7 coated on the detection area 18, and the anti-hemocyanin antibody marked by the fluorescent microspheres 4 coated on the fluorescent pad 14 in advance is directly combined with hemocyanin coated on the quality control area 17, wherein T/C tends to be in a stable ratio; when the sample contains the new coronavirus neutralizing antibody 9, the new coronavirus neutralizing antibody 9 in the sample is combined with the RBD5 marked by the fluorescent microspheres 4 coated on the fluorescent pad 14 in advance, so that the combination of RBD5 and ACE2 protein 7 is blocked, and the anti-hemocyanin antibody marked by the fluorescent microspheres 4 coated on the fluorescent pad 14 in advance is directly combined with hemocyanin coated by the quality control area 17, so that the hemocyanin belongs to specific proteins in arthropods and mollusks hemolymph, the interference on human body samples is avoided, the test result of the quality control area 17 is unchanged, and the T/C value is reduced. Therefore, the content of the new coronavirus neutralizing antibody 9 in the sample is inversely proportional to the T/C value.
The evaluation calculation mode of the new coronavirus neutralizing antibody detection card disclosed by the invention is that the detection result of the detection area 18 is divided by the detection result of the quality control area 17, namely T/C, the detection result of the quality control area 17 is an independent quality control system combined by hemocyanin and an anti-hemocyanin antibody, the detection result is not influenced by the detection substance of the detection area 18, and the detection result of the detection area 18 and the quality control area 17 have no cross influence and are not influenced by the detection result of the quality control area 17. According to the invention, by introducing an independent quality control system, immunoreactions of the quality control area 17 and the detection area 18 are non-homologous and independent from each other, so that mutual influence between the two can be avoided, the quality control area 17 can enable the detection of a novel coronavirus neutralizing antibody to be more accurate by independently marking and coating and participating in result calculation, and the phenomenon that an interferent 11 appears when different samples are tested due to the influence of HAMA (human anti-mouse antibody) in the samples in the traditional detection card and the deviation of a C-line test value is larger by coating the goat anti-mouse antibody 10 in the quality control area is avoided. Meanwhile, the aggregation of the fluorescent substances presented in the quality control region 17 is also a standard for judging whether enough samples exist or not and whether the chromatography process is normal or not, and can also be used as an internal control standard of a new coronavirus neutralizing antibody detection card.
The invention further discloses a preparation method of the novel coronavirus neutralizing antibody detection card, which is described in detail by combining the examples below, but the preparation method is not understood to limit the protection scope of the invention.
Example one
The preparation method of the novel coronavirus neutralizing antibody detection card provided by the embodiment comprises the following steps:
step one, preparing a solution, wherein the solution comprises the following components in percentage by mass:
the sample pad treatment liquid contains: 0.01MOL/L PBS, pH value of 6.0-8.0, 0.1% -1% sucrose, 0.1% -1% BSA, 0.1% -0.5% Tween-20;
the fluorescent pad treatment liquid includes: 0.01MOL/L PBS, pH value of 6.0-8.0, 0.5% -5% cane sugar;
the coating liquid comprises: 0.01MOL/L PBS, pH value of 6.0-8.0, 0.5% -5% sucrose or trehalose;
the coupling liquid comprises: 0.01-0.1 MOL/L, pH value of 5.5-8.0;
the composite solution comprises: buffer solution, 0.05% -0.5% BSA, 0.5% -5% sucrose, 0.5% -5% trehalose;
the above buffers which can be used for the buffer system include phosphate buffer, MES buffer, Tris-HCl buffer, borate buffer, HEPES buffer.
Step two, preparing the fluorescent pad 14: the glass fiber or polyester film is pretreated with the fluorescent pad treatment solution and dried for use.
Step three, sample pad 13 preparation: the glass fiber or polyester film is pretreated with the sample pad treatment solution and dried for use.
Step four, fluorescence labeling:
1. fluorescent microsphere 4-labeled novel coronavirus recombinant protein:
(1) cleaning the microspheres: adding 100ul of fluorescent microspheres 4 into 900ul of activation solution, blowing and uniformly mixing by using a pipette, centrifuging, removing supernatant, adding 1ml of activation solution for later use, wherein the components of the activation solution comprise buffer solution and Tween-20 with the mass concentration of 0.05-0.2%;
(2) activation of microspheres: adding 2.5ul EDC and 2.5ul NHS into the solution (1), activating at room temperature for 15min, centrifuging, removing supernatant, and adding 1ml coupling solution for later use;
(3) coupling microspheres: adding 500ug of novel coronavirus recombinant protein 200 into the solution (2), and reacting at room temperature for 1 h;
(4) and (3) sealing: adding 100ul of blocking solution into the solution in the step (3), reacting for 1h at room temperature, centrifuging and removing supernatant, wherein the components of the blocking solution comprise 10% BSA or 10% sodium caseinate by mass concentration;
(5) redissolving: adding 3-5ml of redissolution into the solution (4) for redissolving.
2. Fluorescent microsphere 4-labeled independent quality control system:
(1) cleaning the microspheres: adding 100ul of fluorescent microspheres 4 into 900ul of activation solution, blowing and uniformly mixing by using a pipette, centrifuging, removing supernatant, adding 1ml of activation solution for later use, wherein the components of the activation solution comprise buffer solution and Tween-20 with the mass concentration of 0.05-0.2%;
(2) activation of microspheres: adding 2.5ul EDC and 2.5ul NHS into the solution (1), activating at room temperature for 15min, centrifuging, removing supernatant, and adding 1ml coupling solution for later use;
(3) coupling microspheres: adding 500ug of hemocyanin 200 to the solution (2) and reacting for 1h at room temperature;
(4) and (3) sealing: adding 100ul of blocking solution into the solution in the step (3), reacting for 1h at room temperature, centrifuging and removing supernatant, wherein the components of the blocking solution comprise 10% BSA or 10% sodium caseinate by mass concentration;
(5) redissolving: adding 3-5ml of redissolution into the solution (4) for redissolving.
3. Mixing the prepared novel coronavirus recombinant protein marked by the fluorescent microsphere 4 with the independent quality control system marked by the fluorescent microsphere 4 according to the ratio of 1:1 to obtain a fluorescent marker for later use.
Step five, ball spraying: the mixed fluorescent marker is sprayed on the fluorescent pad 14 which is pretreated, and the spraying amount is 3-8 ul/cm.
Step six, antibody coating: diluting an anti-hemocyanin antibody to 0.3-1.0mg/ml by using a coating solution, diluting ACE2 protein 7 to 0.8-1.2mg/ml by using the coating solution, coating the anti-hemocyanin antibody on the upper end of a nitrocellulose membrane 15 to form a quality control area 17, and coating the ACE2 protein 7 on the lower end of the nitrocellulose membrane 15 to form a detection area 18.
Step seven, assembling: removing the lining paper of the PVC lining plate 12 during assembly, attaching the nitrocellulose membrane 15 to the middle of the PVC lining plate 12, overlapping the fluorescent pad 14 formed in the fifth step at one end of the nitrocellulose membrane 15 close to the detection area 18, overlapping the sample pad 13 formed in the third step at one end of the fluorescent pad 14 far away from the nitrocellulose membrane 15, overlapping the water absorption pad 16 at one end of the nitrocellulose membrane 15 close to the quality control area 17, and controlling the overlapping part to be 1-3 mm.
Step eight, slitting and loading: the assembled PVC lining plate 12 is cut into test strips of 3-5mm width using a cutting machine, and the test strips are loaded into the corresponding card plugs to obtain finished test card products.
Example two
The technical solution provided by the present invention will be described in detail with reference to another example, in the preparation method of the novel coronavirus neutralizing antibody detection card provided by this example, an independent quality control system not including a fluorescent label is prepared, and this example is used as a comparative example to evaluate the detection result.
Step one, preparing a solution, wherein the solution comprises the following components in percentage by mass:
the sample pad treatment liquid contains: 0.01MOL/L PBS, pH value of 6.0-8.0, 0.1-1% sucrose, 0.1-1% BSA, 0.1-0.5% Tween-20;
the fluorescent pad treatment liquid includes: 0.01MOL/L PBS, pH value of 6.0-8.0, 0.5% -5% cane sugar;
the coating liquid comprises: 0.01MOL/L PBS, pH value of 6.0-8.0, 0.5% -5% sucrose or trehalose;
the coupling liquid comprises: 0.01-0.1 MOL/L, pH value of 5.5-8.0;
the composite solution comprises: buffer solution, 0.05% -0.5% BSA, 0.5% -5% sucrose, 0.5% -5% trehalose;
the above buffers which can be used for the buffer system include phosphate buffer, MES buffer, Tris-HCl buffer, borate buffer, HEPES buffer.
Step two, preparing the fluorescent pad 14: the glass fiber or polyester film is pretreated with the fluorescent pad treatment solution and dried for use.
Step three, sample pad 13 preparation: the glass fiber or polyester film is pretreated with the sample pad treatment solution and dried for use.
Step four, fluorescence labeling of the novel coronavirus recombinant protein:
(1) cleaning the microspheres: adding 100ul of fluorescent microspheres 4 into 900ul of activation solution, blowing and uniformly mixing by using a pipette, centrifuging, removing supernatant, adding 1ml of activation solution for later use, wherein the components of the activation solution comprise buffer solution and Tween-20 with the mass concentration of 0.05-0.2%;
(2) activation of microspheres: adding 2.5ul EDC and 2.5ul NHS into the solution (1), activating at room temperature for 15min, centrifuging, removing supernatant, and adding 1ml coupling solution for later use;
(3) coupling microspheres: adding 500ug of novel coronavirus recombinant protein 200 into the solution (2), and reacting at room temperature for 1 h;
(4) and (3) sealing: adding 100ul of blocking solution into the solution in the step (3), reacting for 1h at room temperature, centrifuging and removing supernatant, wherein the components of the blocking solution comprise 10% BSA or 10% sodium caseinate by mass concentration;
(5) redissolving: adding 3-5ml of redissolution into the solution (4) for redissolution;
(6) ball spraying: the fluorescent-labeled novel coronavirus recombinant protein is sprayed on the pretreated fluorescent pad 14, and the spraying amount is 3-8 ul/cm.
Step five, antibody coating: diluting the goat anti-mouse antibody 10 to 0.3-1.0mg/ml by using a coating solution, diluting the ACE2 protein 7 to 0.8-1.2mg/ml by using the coating solution, coating the goat anti-mouse antibody 10 on the upper end of a nitrocellulose membrane 15 to form a quality control region 17, and coating the ACE2 protein 7 on the lower end of the nitrocellulose membrane 15 to form a detection region 18.
Step six, assembling: removing the lining paper of the PVC lining plate 12 during assembly, attaching the nitrocellulose membrane 15 to the middle of the PVC lining plate 12, overlapping the fluorescent pad 14 formed in the fourth step at one end of the nitrocellulose membrane 15 close to the detection area 18, overlapping the sample pad 13 formed in the third step at one end of the fluorescent pad 14 far away from the nitrocellulose membrane 15, overlapping the water absorption pad 16 at one end of the nitrocellulose membrane 15 close to the quality control area 17, and controlling the overlapping part to be 1-3 mm.
Step seven, slitting and loading: the assembled PVC lining plate 12 is cut into test strips of 3-5mm width using a cutting machine, and the test strips are loaded into the corresponding card plugs to obtain finished test card products.
The accuracy and repeatability of the detection kit for the novel coronavirus neutralizing antibody prepared in the first and second examples are compared and detected.
1. The applicable instrument: fluorescence immunoassay quantitative analysis appearance.
2. A sample to be detected: the serum or plasma collected by clinical standard method comprises 40 cases, wherein 10 cases are not injected with the novel coronavirus vaccine, and 30 cases are 14-28 days after being injected with the novel coronavirus vaccine, and can be stably stored for one week at 2-8 ℃.
3. The detection scheme comprises the following steps:
3.1 accuracy: the detection kits prepared in the first and second embodiments are used to detect 40 clinical samples, compare the samples with ELISA (enzyme-linked immunosorbent assay), and calculate the yin-yang coincidence rate and correlation analysis of the samples with the ELISA.
3.2 repeatability: the assay was repeated 10 times per sample and the CV was calculated.
4. And (3) detection results:
4.1 accuracy test correlation results are shown in table 1, table 2 and figure 3:
TABLE 1 accuracy test results of novel coronavirus neutralizing antibody test kit
TABLE 2 analysis of the rate of coincidence between yin and yang with ELISA method
As can be seen from the detection and analysis results, the independent quality control system product is used for comparison with ELISA, and the compliance rate and the correlation of the independent quality control system are better.
4.2 the results of the reproducibility of the measurements are shown in Table 3:
TABLE 3 repeated detection results of the novel coronavirus neutralizing antibody detection kit
The detection results in table 3 show that the repeatability of the independent quality control system can be controlled within 10%, the CV of the dependent quality control system is larger, and particularly in a low-value area, the CV reaches nearly 20%.
Although the invention has been described in detail above with reference to a general description and specific examples, it will be apparent to one skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed. The above-described embodiments of the present invention do not limit the scope of the present invention. Any modification, equivalent replacement, and improvement made within the spirit and principle of the present invention should be included in the scope of the claims of the present invention.
Claims (12)
1. A novel coronavirus neutralizing antibody detection card is characterized by comprising a PVC liner, and a sample pad, a fluorescence pad, a nitrocellulose membrane and a water absorption pad which are arranged on the PVC liner and sequentially connected;
the fluorescent pad is provided with a novel coronavirus recombinant protein marked by a fluorescent microsphere and an independent quality control system marked by the fluorescent microsphere, wherein the independent quality control system is hemocyanin;
the nitrocellulose membrane is provided with a detection area and a quality control area, the detection area is coated with ACE2 protein, and the quality control area is coated with an anti-hemocyanin antibody;
the hemocyanin of the independent quality control system and the anti-hemocyanin antibody of the quality control area can be interchanged.
2. The novel coronavirus neutralizing antibody detection card according to claim 1, wherein the novel coronavirus recombinant protein is an S protein receptor binding domain.
3. The novel coronavirus neutralization antibody detection card according to claim 1, wherein the nitrocellulose membrane is attached to the middle part of the PVC pad, the fluorescent pad and the water absorption pad are respectively lapped on the upper surfaces of two ends of the nitrocellulose membrane, the sample pad is lapped on the upper surface of the fluorescent pad, and the lapping and overlapping parts are all arranged at 1-3 mm.
4. The detection card for neutralizing antibody against coronavirus as claimed in claim 1, wherein the fluorescent microsphere has a solid content of 0.1-5% and a particle size of 100-300 nm.
5. The novel coronavirus neutralizing antibody detection card according to claim 1, wherein the PVC pad is set to have a width of 3-5 mm.
6. A preparation method of a novel coronavirus neutralizing antibody detection card is characterized by comprising the following steps:
the method comprises the following steps: preparing a fluorescent pad and a sample pad, pretreating and drying the glass fiber or polyester film for later use;
step two: fluorescent labeling of novel coronavirus recombinant proteins;
step three: fluorescence labeling of the independent quality control system;
step four: uniformly mixing the novel coronavirus recombinant protein fluorescently labeled in the step two and the independent quality control system fluorescently labeled in the step three according to the proportion of 1:1 to obtain a fluorescent marker for later use;
step five: spraying the fluorescent marker of the step four on the fluorescent pad;
step six: respectively diluting an anti-hemocyanin antibody and ACE2 protein, and then coating the diluted anti-hemocyanin antibody and ACE2 protein on a nitrocellulose membrane to respectively form a quality control area and a detection area;
step seven: sticking the nitrocellulose plastic film formed in the sixth step to the middle of a PVC lining plate, lapping the fluorescent pad formed in the fifth step on one end of the nitrocellulose plastic film, lapping the sample pad formed in the first step on one end, far away from the nitrocellulose plastic film, of the fluorescent pad, and lapping the water absorption pad on the other end of the nitrocellulose plastic film;
step eight: and (6) cutting the test strip.
7. The method for preparing the detection card of neutralizing antibody against coronavirus of claim 6, wherein the step two, the fluorescence labeling of recombinant protein of coronavirus comprises the following steps:
the first process is as follows: cleaning microspheres, adding 100ul of fluorescent microspheres into 900ul of activating solution, blowing and uniformly mixing by using a pipette, centrifuging, removing supernatant, and adding 1ml of activating solution for later use;
and a second process: activating microspheres, adding 2.5ul EDC and 2.5ul NHS into the solution in the first process, activating at room temperature for 15min, centrifuging, removing supernatant, and adding 1ml of coupling solution for later use;
and a third process: coupling microspheres, adding 500ug of the novel coronavirus recombinant protein 200 into the solution in the process II, and reacting at room temperature for 1 h;
and (4) a fourth process: sealing, adding 100ul of sealing liquid into the solution in the third process, reacting for 1h at room temperature, centrifuging and removing supernatant;
and a fifth process: and (4) redissolving, namely adding 3-5ml of redissolving solution into the solution in the process four for redissolving.
8. The method for preparing the novel coronavirus neutralizing antibody detection card according to claim 6, wherein in the third step, the fluorescence labeling independent quality control system comprises the following procedures:
the first process is as follows: cleaning microspheres, adding 100ul of fluorescent microspheres into 900ul of activating solution, blowing and uniformly mixing by using a pipette, centrifuging, removing supernatant, and adding 1ml of activating solution for later use;
and a second process: activating microspheres, adding 2.5ul EDC and 2.5ul NHS into the solution in the first process, activating at room temperature for 15min, centrifuging, removing supernatant, and adding 1ml of coupling solution for later use;
and a third process: coupling microspheres, adding 500ug of hemocyanin 200 into the solution in the process II, and reacting for 1h at room temperature;
and (4) a fourth process: sealing, adding 100ul of sealing liquid into the solution in the third process, reacting for 1h at room temperature, centrifuging and removing supernatant;
and a fifth process: and (4) redissolving, namely adding 3-5ml of redissolving solution into the solution in the process four for redissolving.
9. The method for preparing a novel coronavirus neutralizing antibody detection card according to claim 6, wherein in the fifth step, the fluorescent marker is sprayed on the fluorescent pad in an amount of 3-8 ul/cm.
10. The method for preparing a novel coronavirus neutralizing antibody detection card according to claim 6, wherein in the sixth step, the anti-hemocyanin antibody and the ACE2 protein are diluted by coating solutions respectively, the anti-hemocyanin antibody is diluted to 0.3-1.0mg/ml, and the ACE2 protein is diluted to 0.8-1.2 mg/ml.
11. A novel coronavirus neutralizing antibody detection kit comprising the novel coronavirus neutralizing antibody detection card according to any one of claims 1 to 5.
12. The novel coronavirus neutralizing antibody assay kit of claim 11, further comprising a sample diluent comprising: Tris-HCl buffer solution with the pH value of 8.0-9.5, BSA with the mass concentration of 0.1-1% and Proclin300 with the mass concentration of 0.1-0.5%.
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